Lactobacillus reuteri (LR) has beneficial effects in several human diseases. Necrotizing enterocolitis (NEC) is the most common severe gastrointestinal condition affecting 7% of premature infants. Our preliminary studies demonstrated in neonatal mice with experimental NEC that LR feeding reduces mortality and intestinal inflammation, while increasing intestinal mucosal regulatory T cells (Tregs), cells with anti-inflammatory properties. Specific Aims: 1. To determine whether LR facilitates the generation of tolerogenic dendritic cells (DCs) via bacterial recognition receptors on DC (sentinel cells) called Toll like receptors (TLRs). Gut inflammation is believed to be sustained by DC interaction with mucosal helper T cells (Th1 and Th17) and reduced by Tregs. We will measure NEC severity, response to LR, percentage of Tregs, Th1, and Th17 effectors in mice genetically deficient in TLRs. 2. To elucidate if LR-conditioned Tregs will be more efficient to suppress Th1/Th17 effectors in the inflamed gut, we will adoptively transfer Tregs bearing congenic markers from LR-fed mice to newborn mice undergoing NEC to determine if exogenous Tregs protect. 3. To determine whether LR is capable of inducing Tregs (iTregs) in the intestinal mucosa when natural Tregs (nTreg) are depleted, we will deplete nTregs by anti-CD25 antibody and determine if LR enhances iTreg development and remains capable of reducing intestinal inflammation. These aims will provide novel insights into mechanisms of Lactobacillus reuteri regulation of neonatal intestinal inflammation. Results will facilitate the selection of biomarkers to follow the evolution of NEC and to compare the potency of different probiotics in the human infant.